Despite improvements in the treatment of Non Small Cell Lung Cancer (NSCLC hereafter), current approaches for patients with more advanced disease such as chemotherapy and radiotherapy have had little impact on patient survival. See “Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials,” Non-small Cell Lung Cancer Collaborative Group Bmj, 311:899-909 (1995); Socinski, et al., Chest, 123:226 S-243, (2003); Dubey and Schiller, Hematol Oncol Clin North Am, 18:101-114 (2004). An improvement in the understanding of molecular processes involved in pulmonary carcinogenesis has led to new treatment options with targeted small molecules and vaccines demonstrating encouraging potential. See Shepherd, et al., N Engl J Med, 353:123-132 (2005); Tsao, et al., N Engl J Med, 353:133-144 (2005); Zhang, et al., Cancer Res, 66: 9736-9743 (2006); O'Mahony, et al., J Clin Oncol, 23: 9022-9028 (2005); Hirschowitz, et al., J Clin Oncol, 22:2808-2815 (2004).
The heterogeneity of clinical outcome in lung cancer patients with similar stage disease, spontaneous regression of tumors and improved survival amongst patients with tumor-infiltrating lymphocytes and those who develop empyemas provide evidence that immune responses may influence outcome in NSCLC patients. See Shankaran, et al., Nature, 410:1107-1111 (2001); Ruckdeschel, et al., N Engl J Med, 287:1013-1017 (1972); Wei and Hang, Immunol Invest, 18:1095-1105 (1989). Utilizing the immune system to target lung carcinoma is thus an approach that should be further explored.
Cancer-testis antigens, or “CTAs”, are members of a family of antigens expressed in cancer, with minimal expression in non-cancerous tissues. See, e.g., Tureci, et al., Proc. Natl. Acad. Sci. USA, 95:5211-5216 (1998); Gure, et al., Int. J. Cancer, 72:965-971 (1997); U.S. Pat. No. 6,140,050, all of which are incorporated by reference in their entireties. Many CTAs are immunogenic (Simpson, et al., Nat Rev Cancer, 5:615-625, (2005)), characterized by restricted expression in testis but aberrant expression in a variety of cancer types including NSCLC. See Scanlan, et al., Cancer Immun, 4:1 (2004). These properties render them attractive candidates for cancer vaccines. Indeed vaccination with either the full length recombinant CTA NY-ESO-1 protein, or CD4- or CD8-restricted peptide epitopes, enhances anti-NY-ESO-1 reactivity. See Maraskovsky, et al., Clin Cancer Res, 10: 2879-2890 (2004); Davis, et al., Proc Natl Acad Sci USA, 101: 10697-10702 (2004). Tumor regression has been achieved in some cases. See Jager, et al., Proc Natl Acad Sci USA, 97: 12198-12203 (2000).
Embryogenesis involves a de-programming or erasure of the epigenetic information governing differentiated cell behavior, thus returning the cell to the proliferative, undifferentiated, stem cell state. See Monk, et al., Development, 99: 371-382 (1987); Mayer, et al., Nature, 403: 501-502 (2000); Reik, et al., Science, 293: 1089-1093 (2001). By the blastocyst inner cell mass (ICM) and the PGC stages, the embryonic cells are totipotent stem cells (cells that can give rise to any and all adult cell types) capable of giving rise to immortal cell lines in vitro and teratomas in vivo. Embryonic genes which are active at this stage may be associated with similar properties of deprogramming, maintenance of the undifferentiated cell state, proliferation, invasiveness and indefinite growth of cancer cells. The identification of several Embryo-Cancer transcripts that are expressed in human pre-implantation embryos, absent in normal differentiated somatic tissues but re-expressed in tumor tissue, supports this hypothesis. See Monk, et al., Oncogene, 20:8085-8091 (2001), incorporated by reference in its entirety.
Monk, et al. described finding a portion of the gene referred to as Embryo-Cancer Sequence A (ECSA) in human pre-implantation embryos, primordial germ cells (PGCs) and several somatic tumors. This gene was subsequently renamed and entered into gene databases as Developmental Pluripotency Associated-2 (DPPA2). See Genbank Accession Number NM—138815, incorporated by reference and set forth at SEQ ID NO: 9. This gene maps to chromosome 3q13 over 8 exons and encodes a protein product of 297 amino acids. The primary protein structure contains a SAP motif and localizes to the nucleus.
Using a bioinformatics approach in the mouse, DPPA2, was found to have an expression pattern similar to OCT 3/4 (Bortvin, et al., Development, 130: 1673-1680 (2003)), one of several molecules including NANOG and SOX2 (Boiani, et al., Nature Reviews Molecular Cell Biology, 6: 872-881 (2005)), that are characteristically present in pluripotent stem cells (primordial cells that may still differentiate into various specialized types of tissue elements). The gene encoding DPPA4, another molecule which shares this expression pattern, lies 16 kB upstream of DPPA2 and encodes a closely related SAP domain. See Bortvin, et al., supra; Maldonado-Saldivia, et al., Stem Cells, 25: 19-28 (2007). These domains are thought to be involved in RNA metabolism and in the organization of nuclear architecture, suggesting that both may be involved in controlling cellular plasticity. See Aravind, et al., Trends Biochem Sci, 25: 112-114 (2000). DPPA2 and DPPA4 are both found exclusively in pluripotent cells; however a recent study involving murine embryonic stem cells suggests that DPPA4 may not be critical in maintenance of the pluripotent phenotype, as its over-expression did not inhibit cellular differentiation. See Ivanova, et al., Nature, 442: 533-538 (2006). Similarly, other markers including STELLAR, LEFTY2, and GDF3 are also expressed in pluripotent cells, although their roles are less well defined.
Germ cells also express a number of these molecules so it is not surprising that NANOG, OCT3/4, GDF3 and STELLAR have been found in germ cell tumors. See Chambers, et al., Oncogene 23: 7150-7160 (2004); Clark, et al., Stem Cells 22: 169-179 (2004); Hart, et al., Cancer 104: 2092-2098 (2005). In contrast, a few publications (limited to breast cancer (See Jin, et al., Int J Cancer 81: 104-112 (1999); Mongan, et al., Mol Carcinog (2006); Ponti, et al., Cancer Res 65: 5506-5511 (2005); Ezeh, et al., Cancer 104: 2255-2265 (2005) and bladder cancer (See Atlasi, et al., Int J of Cancer; 120: 1598-1602 (2007)) implicate these molecules in tumors arising from somatic cells. Their expression in somatic tumors may underpin biological functions that cancers share with embryonic cells, primordial germ cells and possibly adult stem cells, such as self renewal and proliferative potential. Suppression of differentiation is a hallmark of pluripotent embryonic cells, and although cancer cells may not necessarily have the capacity to proliferate along multiple differentiated lineages, loss of differentiation is a common feature.
Immunogenicity of these molecules in cancer patients has previously only been investigated for SOX2. Serum antibodies have been reported against SOX2 in meningioma and small cell lung cancer (SCLC) (Comtesse, et al., PNAS 102: 9601-9606 (2005); Gure, et al., PNAS; 97: 4198-4203 (2000)), although it is unclear how these responses arose since SOX2 expression was not documented in the meningiomas and expression in the SCLC patients was not investigated. A recent publication has also demonstrated SOX2 antibodies in patients with monoclonal gammopathy of undetermined significance (MGUS) in which the SOX2 defined the clonogenic cells. See Spisek, et al., J. Exp. Med. 2007 jem.20062387. In this study, the presence of serum antibody was also associated with T-cell responses and improved clinical outcome, although the numbers reported were small.
The experiments described herein explore the relationship between the embryo-associated antigen ECSA/DPPA2 and the germ-cell associated CT antigens (CTAs). The CTAs are not known to be markers of pluripotent stem cells although their expression in germ cells and various cancers, suggests a role in primitive cell populations. A recent review of the CTAs by Simpson et al provides a conceptual framework which links cancer with germ cell development. See Simpson, et al., Nat Rev Cancer; 5: 615-625 (2005). The authors suggest that the epigenetic dysregulation and subsequent de-repression of germ cell programs that would normally be silenced in somatic cells may contribute to de-differentiation and the subsequent malignant phenotype in cancer cells.
ECSA/DPPA2 can best be classified as an “Embryo-Cancer Antigen”, as reflected by its association with embryogenesis rather than gametogenesis. Supporting this view is that ECSA/DPPA2 is expressed in pluripotent embryonic cells, human embryonic stem cell (hESC) lines as well as PGCs. In contrast, CTAs have not been reported to be expressed in hESCs or pre-implantation embryos. Secondly, ECSA/DPPA2 is co-expressed with other well defined markers of pluripotent stem cells including OCT3/4 and NANOG. See Monk, et al., supra; Maldonado-Saldivia, et al., supra. The association between these molecules and ECSA/DPPA2 is closely linked whereas a similar relationship with the CTAs has not been shown. Thus, ECSA/DPPA2 has unique associations since it belongs to a group of molecules that is implicated in pluripotent stem cells, on the one hand, and shares expression patterns in common with CTAs on the other.